Ruxolitinib-based senomorphic therapy mitigates cardiomyocyte senescence in septic cardiomyopathy by inhibiting the JAK2/STAT3 signaling pathway

Background: Cellular senescence has emerged as a pivotal focus in cardiovascular research. This study investigates the previously unrecognized role of cellular senescence in septic cardiomyopathy (SCM) and evaluates senomorphic therapy using ruxolitinib (Rux) as a potential treatment option. Methods: We employed lipopolysaccharide (LPS)-induced neonatal rat cardiomyocytes (NRCMs) and two mouse models—LPS-induced and cecal ligation and puncture (CLP)-induced SCM models—to assess Rux's effects. RNA sequencing, western blotting (WB), quantitative polymerase chain reaction (qPCR), immunofluorescence, immunohistochemistry, senescence-associated β-galactosidase (SA-β-gal) assay, and other techniques were utilized to investigate underlying mechanisms. Results: Senescence-associated secretory phenotype (SASP) and cellular senescence markers were markedly elevated in LPS-induced NRCMs and SCM animal models, confirmed by the SA-β-gal assay. Rux treatment attenuated SASP in vitro and in vivo, alongside downregulation of senescence markers. Moreover, Rux-based senomorphic therapy mitigated mitochondrial-mediated apoptosis, improved cardiac function in SCM mice, restored the balance of antioxidant system, and reduced reactive oxygen species (ROS) levels. Rux treatment restored mitochondrial membrane potential, mitigated mitochondrial morphological damage, and upregulated mitochondrial complex-related gene expression, thereby enhancing mitochondrial function. Additionally, Rux treatment ameliorated SCM-induced mitochondrial dynamic dysfunction and endoplasmic reticulum stress. Mechanistically, Rux inhibited JAK2-STAT3 signaling activation both in vitro and in vivo. Notably, low-dose Rux and ABT263 showed comparable efficacy in mitigating SCM. Conclusions: This study highlighted the potential significance of cellular senescence in SCM pathogenesis and suggested Rux-based senomorphic therapy as a promising therapeutic approach for SCM.

#G0750, Sigma-Aldrich, USA) was used at a concentration of 10 g/L.The grouping was similar to that described above.The Rux therapy began 6 hours after D-gal until 24 hours.

Figure
Figure S1 Bioinformatics analysis based on human heart samples obtained from 11 donors whose hearts did not fail and 20 patients with SCM using GSE79962 dataset (A, B) Relative expression levels of senescence markers, including P21 and P53, between nonfailing hearts and septic hearts.(C) Relative expression levels of SASP-related genes, including IL-1R1, IL-6R, IL-18R1, TNFα-IP1, CCL2, EDN1, TGFβ1, CXCL8, and MMP-1, were compared betweennonfailing hearts and septic hearts (D) KEGG analysis revealed that cellular senescence and JAK-STAT signaling pathway were changed between nonfailing hearts and septic hearts (E) GSEA analysis revealed that cellular senescence was induced in septic hearts compared with non-failing

Figure S2
Figure S2 Rux alleviates cellular senescence in H 2 O 2 -and D-gal induced cell models.(A)Representative Western blot bands and quantitative analysis of P16 and P53 in NRCMs treated with Rux or not post H2O2 stimulation compared with the control group.N = 3. (B) The mRNA levels of P16, P21 and P53 in NRCMs treated with Rux or not post H2O2 stimulation compared with the control group.N = 4. (C) The mRNA levels of SASP-related genes were detected using qRT-PCR in NRCMs treated with Rux or not post H2O2 stimulation, including IL-6, TNF-α, CXCL1, CXCL3, CXCL10, CCL2, and EDN3.N = 4. (D, E) Representative Western blot bands and quantitative analysis of P16 in NRCMs treated with Rux or not post D-gal stimulation compared with the control group.N = 3. (F) The mRNA levels of P16 and P21 in NRCMs treated with Rux or not post D-gal stimulation compared with the control group.N = 3.Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.Con, control; Rux, ruxolitinib; D-gal, Dgalactose.

Figure
Figure S5 Verification of the downstream genes of the JAK2/STAT3 pathway.(A)The mRNA levels of PSMB8, PSMB9, TAP1 and IRF7 were detected using qRT-PCR in NRCMs treated with Rux or not post LPS stimulation compared with the control group.N = 4. (B) The mRNA levels of PSMB8, PSMB9, TAP1 and IRF7 were detected using qRT-PCR in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group.N = 5.Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Figure
Figure S6 Rux improves cardiac function in septic heart disease partly through cellular senescence.(A) Schematic diagram of the experiments.(B, C) Representative Western blot bands and quantitative analysis of P16 in mouse hearts transfected with adenovirus P16 or GFP treated with Rux or not post LPS injection.N = 3. (D) The mRNA levels of P21 in mouse hearts transfected with adenovirus P16 or GFP treated with Rux or not post LPS injection.N = 3. (E) Cardiac function indices were measured by echocardiography in mouse hearts transfected with adenovirus P16 or GFP treated with Rux or not post LPS injection.N = 5. (F) Representative images of SA-β-gal staining in mouse hearts transfected with adenovirus P16 or GFP treated with Rux or not post LPS injection.Scale bar = 50 μm.Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.Adv, adenovirus; LPS, Lipopolysaccharide; Rux, ruxolitinib.

Table S2 .
The primer sequences of qRT-PCR.

Supplementary Materials H 2 O 2 -and D-gal-induced cell models 400μM
H 2 O 2 was used to stimulate NRCMs for 3 hours to construct a cell model different from that of LPS.The study included a control group of NRCMs, a group of NRCMs treated with 1μM of Rux, a group of H 2 O 2 -induced NRCMs, and a group of H 2 O 2 -induced NRCMs treated with 1μM Rux.The Rux therapy began 3 hours after H 2 O 2 stimulation until 12 hours.D-galactose (D-gal;